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2016 Pharma Tour

Characterization of mAbs Using RP Separations and High Resolution Accurate Mass (HRAM) Mass Spectrometry

2

Antibody Therapeutics – An Analytical Challenge

Deamidation (+1 Da)

Oxidation (+16 Da)

Disulfide bond scrambling

Sequence truncation

Change in glycosylation

Low abundance host cell proteins

Low abundance sequence variants

Aggregation, protein complex

Drug Antibody Ratio’s

Linker measurements

Bound vs unbound drug

etc

• to ensure proper biomanufacturing of the therapeutics • to ensure critical quality attributes (CQA’s) of a product are identified

Modifications, impurities, immunogenicity, efficacy, DMPK • to ensure these CQA’s can be routinely measured

Mass spectrometers, separations, software, reagents

Characterization

Monoclonal antibody (mAb)

Antibody drug

conjugate (ADC)

3

MS Tools for Major Biopharma Characterization Workflows

For all the routine characterization workflows: intact/top-down, bottom-up,

glycan, etc. qualitive and quantitative.

Native MS to analyze large protein complex while

preserving non-covalent interactions: antibody-antigen

complex, ADCs, antibody mixtures, etc.

From routine to the most challenging tasks. The most

advanced technology that provides the most

comprehensive solutions.

4

The Orbitrap™ in Hybrid and Non-Hybrid Systems

LTQ Orbitrap XL™ Orbitrap Elite™

Exactive Plus EMR Q Exactive Q Exactive Plus Q Exactive HF

Source Orbitrap Mass Analyzer

Bent Flatapole

Z lens

Quadrupole C-Trap

C-Trap Entrance

Lens HCD Collision Cell

Orbitrap Fusion Tribrid Orbitrap Fusion Lumos™ Tribrid

5

time [ms]

Am

plitu

de

The Orbitrap Principle

frequency

FT

m/z

Transient

6

Vanquish Platform

• Improved retention time precision • Biocompatible by default • Increased sample capacity • Intelligent sample Pre-compression • Improved sample cooling • Multiple heating modes • Active and passive pre-heating • 4 Detection options • Enhanced LC/MS control with Sii and Chromeleon • Viper™-based, tool-free fluidic connections

Vanquish Flex

• 1000 bar compatible system • Quaternary pump (LPG) • Medium Gradient Delay Volume

Separations based on chemistry

Vanquish

• 1500 bar compatible system • Binary pump (HPG) • Ultra low Gradient Delay Volume

The Thermo Scientific™ Vanquish™ UHPLC System… A Perfect Front-end!

Separations based on power

LCMS Control

7

Antibody Characterization Roadmap

Released glycans

Intact mAb Deglycosylated mAb

PNGaseF

DTT, TCEP

Trypsin, LysC, etc.

IdeS, Papain, w/ DTT, TCEP

Reduced Subunits

Reduced Digested Subdomains

Peptide Mapping PNGaseF

8

Agenda

Major Characterization Workflows

9

Intact mass Analysis

Output

• Identify glycoforms using the intact mass.

• Identify Antibody Drug Conjugates (ADC).

• Confident deconvoluted molecule weight.

(LC: in native or denaturing conditions)

LC-MS IgG

(150 kDa)

2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.Time (min)

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

3.57

3.82 4.043.50 4.45 4.73 5.05 5.33.232.922.462.23

1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

2907.25

2797.59 3025.87

3088.892745.82

2695.90

2968.59

3154.57

2647.78 3223.152601.35

3294.722556.54

2513.21 3369.59

2430.84 3447.922353.71

3529.932246.72 3616.002149.04 3801.46 3901.522003.931899.12

10

BioPharma Finder™ 1.0

One software package for

• Protein Sequence Management

• Peptide Mapping • Intact Analysis

11

BPF 1.0 – Protein Sequence Manager

Protein Sequence Manager

Saves time

• Starting point for the software.

• Database for storing protein sequences similar to Proteome Discoverer.

• Both peptide mapping and intact analysis workflow choose protein sequence from this central location.

Easy to use

• Import FASTA file or paste the sequence into a text box.

• Define multiple chains (e.g. two light chains, two heavy chains).

• Define disulfide bonds (information used for intact analysis)

• Choose fixed modifications

• Select variable modifications and list of possible glycosylation structures.

• User definable default modification list.

12

BPF 1.0 – Intact Protein Analysis

Intact Protein Analysis • Determine profiles of intact masses

• Automated processing, simplified results and improved quantitation

• Sliding window algorithm for improved ADC analyses and complex data sets

• Target protein sequence matching – identifies n-linked glycoylations and other common modifications.

• Native MS • Ion trap support (ReSpect™ deconvolution

only) Sliding Window Deconvolution • Produces a single deconvoluted spectrum of

all components in the sample regardless of elution time or profile

• Eliminates the need for the user to define the source spectra for deconvolution.

• Works for both simple and highly complex protein mixtures

• Removes the challenges of chromatographic peak picking

• Works with Xtract™ and ReSpect deconvolution

13

Intact Protein Analysis

• A fast 4 minutes desalting method for high throughput glycan characterization.

• Intact mass and the relative glycoform abundance of a mAb within 5 minutes.

• In-depth characterization for glycoforms detection below 1% relative intensity.

• Broadly applicable to other mAbs and similar biopharmaceutical compounds.

2000 2500 3000 3500 4000 m/z

2727.6373 z=54

2539.6085 2945.7809

2454.9930 3133.7244

2375.8333 3272.9270

3506.6994 2201.0665

2640 2660 2680 2700 2720 2740 2760 m/z

2727.6373 z=54

2678.0783 z=55 2630.2572

z=56

2736.6398 2686.9898 2639.0313

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Time (min)

2.40

B

A

C

0

1

2

3

4

Rel

ativ

e In

tens

ity

147237.98 147400.22147076.45 147560.95 147724.64

147854.70

146861.92

146701.11 148145.02148014.19 148305.77

146623.00146931.52

146600 146800 147000 147200 147400 147600 147800 148000 148200 148400Mass

NL:

1.31E9

NL:

1.31E9

G0F/G0F G0F/G1F G0F/G2F or (G1F)2 G1F/G2F G2F/G2F

(G1F/G2F)SA1

(G2F/G2F)SA1

(G1F/G2F)SA2

(G2F/G2F)SA2

G0/G0F

Man5/G1

Man5/G0 (Man5)2

D

Download Application Note 21465: Fast online desalting of mAbs using a reversed phase desalting cartridge for LC-MS analysis

Carbohydrate structures commonly observed on antibodies.

For more separation power use MAbPac-RP

14

MS of Intact Lysine-linked ADC

2860 2880 2900 2920

2867.3693

2886.0424

2848.9049 2904.9884

2924.6023

0

1 2 3

4

5 6 7

0 1 2

3 4

5

DAR: 1.5

DAR: 2.5

RE

LATI

VE

INTE

NS

ITY

MASS

Q Exactive, Resolution Setting = 17.5 K

D0

ΔM

D1

ΔM

D3

D4

D7

D6

D5ΔMΔM ΔM

ΔM

ΔM

DAR=3.23

0

100

Abun

danc

e

mass 145000 146000 147000 148000 149000 150000 151000 152000

D2 D3

DAR: 3.23

15

Separation of ADC

0 4 8 12 16 20

0

10

20

30

40

Abs

orba

nce

(mA

U)

DAR 0

DAR 2

DAR 4

DAR 6

DAR 8

by Hydrophobic Interaction (HIC) by Reversed Phase for direct coupling with MS

MMAE MMAE

MMAE MMAE

UV

-1000

0

1000

2000

3000

4000

5000

uAU

2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5

Cys-linked ADC mimic

MAbPac RP (4 µm) 2.1 × 50 mm

MAbPacHIC-Butyl (5 µm) 4.6 ×100 mm

MAbPac HIC-Butyl (polymer, non-porous)

MAbPac HIC-10 (polyamide, silica, 1000Å)

MAbPac HIC-20 (alkylamide, silica, 1000Å)

Phenyl RP chemistry super-macroporous polymer particle (1500Å) - ultra-low carry-over - pH 0-14 stabile - up to 110°C

Applications: ADC, mAb Fragments and Aggregates Oxidation and Disulfide bridge variants

min min

16

Only for these 3 isomers the LC and HC are still linked through covalent bonds.

MS of Intact Cysteine-linked ADC

DAR 0

Inter-chain disulfide bond

Linker + drug

DAR 2 DAR 6 DAR 8 DAR 4

Cysteine linked ADC requires native MS for intact analysis

17

Native MS using Exactive Plus EMR

• An ESI compatible volatile solution with near neutral pH is used to prepare protein samples. e.g. 50 mM ammonium acetate pH 6.9

• Under these conditions, the ionized proteins carry fewer charges than those produced by ESI in acidic, denatured condition.

• Therefore, in native MS, protein ions are detected at a significantly higher m/z range (>5000 -10,000 m/z for antibody complexes) than conventional MS in acidic condition.

• Extended mass range up to m/z 20,000

• Unmatched Desolvation using in-source CID and in HCD cell

• Improved high mass transmission for large protein assemblies

3000 4000 5000 6000 7000m/z

0

20

40

60

80

1000

20

40

60

80

100

Relati

ve Ab

unda

nce

3276.463350.953071.72

3428.83

3510.48

2948.91

3596.112835.51

3685.953780.49

2730.54

3984.74

6410.47

6701.82

5897.61

7020.98

5670.63

mAb, Acidic pH, Denatured

mAb, Neutral pH, Native

18

Native MS of an ADC Brentuximab Vedotin

Exactive Plus EMR, 35K

DAR4

DAR2

DAR6

DAR8 DAR0

146000 144000 Mass (Da) 150000 148000 154000 152000 158000 156000

+2634.2 Da

+2635.4 Da

+2634.3 Da +2635.1 Da

145893.6 “ 2.2 Da

148527.8 “ 0.8 Da

151163.2 “ 0.9 Da

153797.5 “ 0.7 Da

156432.6 “ 1.8 Da

DAR8

= 3.8

∑

∑ =

8

0

8

0

n

n

DAR

DAR

A

nA DAR = 4.2

Raw spectrum Deconvoluted spectrum

19

Agenda

Major Characterization Workflows

20

Reduced Rituximab

Light chain acquired with 140k Resolution

Heavy chain acquired with 17k Resolution

10 11 12 13 14 15 16 17 18 19 20 Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

16.96

14.57

x5

1000 1500 2000 2500 3000 m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

2304.66 2560.31

1213.34 1536.83 1920.51

1152.82 1646.32 2095.17

1098.02 2880.87

960.89

1780.49

2661.69

1500 2000 2500 m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

1810.86 1950.07

1690.16 2028.00

1584.58

1536.59 2112.45

2304.44

2414.12

2668.24 2816.35

2982.00

m/z 2020 2040

2028.00 2021.53

2034.56

21

Rapid Subdomain Analysis of NIST mAb

Matched Sequence Matched Delta Mass (ppm)

Relative Abundance

Fd 1.05 100.0000 LC 2.50 86.4514

Fc;1xA2G1F 2.34 34.9027

Fc;1xA2G0F 2.47 34.8168

Fc;1xA2G2F 2.48 3.6534 0

10

20

30

40

50

60

70

80

90

100

R e l

a t i v

e I n

t e n s

i t y

23000 23500 24000 24500 25000 25500 Mass

25668.840 NIST mAb Fd

25216.495 NIST mAb Fc

1xA2G0F

25378.545 NIST mAb Fc

1xA2G1F

25540.602 NIST mAb Fc

1xA2G2F 23109.298 NIST mAb LC

1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z

0 10 20 30 40 50 60 70 80 90

100

Rel

ativ

e Ab

unda

nce

1284.5 1285.0 1285.5 1286.0 1286.5 m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

z=20

z=18 z=20 z=20 z=20 z=18

z=18

z=18 z=20

z=18 z=20

z=18 z=20 z=18

z=18

z=20 z=18 z=20 z=18

XtractTM deconvolution

Fusion Lumos R=240,000

22

Agenda

Major Characterization Workflows

23

• Isolation window • AGC target for precursor ions and reagent

• Reaction time

• Supplemental energy

24

ETD settings: 300 Da isolation window, 3E5 precursor, 7E5 reagent, 10 ms reaction time

Due to the complexity of the spectra in top-down analysis, high resolution is required

25

Top-Down Sequencing D

econ

volu

tion

Matching

26

Light Chain

Fc Fd

High sequence coverage for the light chain, Fc and Fd were obtained from the combined ETD and EThcD experiments.

27

Improved Coverage For Top Down IgG Analysis

Comprehensive Sequence Map

• Reduced IgG, infusion at 3 ul/min

• Individual charges states of both proteins were isolated and fragmented with CID, ETD and HCD

• 240,000 resolution

• Improved sequence coverage with Advanced Vacuum Technology and ETD HD

Light Chain (LC)

Heavy Chain (HC)

c z b y

91%

63% OT FUSION OTF LUMOS GAIN

LC 77% 91% 18%

HC 46% 63% 37%

28

Closing

Acknowledgements

• Stephane Houel

• Terry Zhang

• Aaron Bailey

• Michael Blank

• Jonathan Josephs

• Martin Samonig

• Kai Scheffler

• Yue Xuan

• Eugen Damoc

• Remco Swart

• Jennifer Sutton

• Seema Sharma

• David Horn

• Shanhua Lin

• Mark Sanders

• Ken Miller